精子冷冻保存在大熊猫种质资源保护和人工繁育中具有重要的作用。但是大熊猫冷冻精液人工授精的产仔率低下，导致冷冻精液利用效率极差，原因在于冷冻对大熊猫精子的损伤及作用机制尚不明确。miRNA在精子发生和成熟过程中具有重要的调控作用，并能影响受精后早期胚胎的发育。CryomiRs是耐寒性昆虫和冬眠动物中一类与抗冷/抗冻相关的miRNAs。我们前期研究发现，冷冻能够诱导精子miRNA表达的变化，推测miRNA及靶基因可能参与调控精子重要的生理生化过程，影响精子的冻后质量和受精效果。因此，本项目拟采用miRNA高通量测序、流式细胞分析和miRNA干扰等技术，探讨冷冻过程中大熊猫精子CryomiRs表达规律及其靶基因作用的信号途径，揭示miRNA调控大熊猫精子冷冻保存效果的作用机制，从新的角度阐明大熊猫冷冻精液人工授精效率低下的原因，为优化和改善大熊猫精子冷冻稀释液提供新的思路。

Spermatozoa cryopreservation plays an important role in the long-term storage of giant panda genetic resources and artificial breeding. However, the unidentified action and cryoinjury mechanism result in low birth rate and poor utilization efficiency using artificial insemination with frozen-thawed semen of giant panda. microRNA (miRNA) can regulate spermatogenesis, maturation process of spermatozoa, and also effect the early embryonic development after fertilization. CryomiRs, a cold-associated family of miRNAs, are mainly involved in cold or freezing tolerance in some insects and hibernating mammals under extreme environmental stresses through the depression of metabolic rate and other physiological and biochemical processes. Our previous study indicated that cryopreservation can induce miRNA expression changes and probably modulate metabolic-related physiological and biochemical processes of spermatozoa. It was hypothesized that miRNAs and their target genes may participate in the regulation of important physiological and biochemical process of spermatozoa, and then affect post-thawed semen quality and its fertilization effects of giant panda. To identify this hypothesis, this project aims to detect the cryomiRs expression and the signal pathways of their target genes using high-throughput miRNA sequencing, flow cytometry and RNA interference techniques, and to explore the mechanism of miRNAs modulating the cryopreservation effects of giant panda frozen semen, and try to explain, from a new point of view, the reason of low efficiency of artificial insemination with frozen-thawed semen and provide a new idea for the optimization and improvement of the freezing extenders of giant panda spermatozoa.